Liposomal antineoplastic drugs and uses thereof

ABSTRACT

This invention relates to liposomal antineoplastic agents (e.g., camptothecin) compositions and methods of using such compositions for treating neoplasia and for inhibiting angiogenesis. The compositions and methods are useful for modulating the plasma circulation half-life of an active agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. Provisional Application No.60/215,556, filed Jun. 30, 2000, entitled “Liposomal Camptothecins andUses Thereof,” and U.S. Provisional Application No. 60/264,616, filedJan. 26, 2001, entitled “Liposomal Antineoplastic Drugs and UsesThereof,” both of which are incorporated herein by reference in theirentireties for all purposes. U.S. patent application Ser. No.09/896,811, filed Jun. 29, 2001, entitled “Liposomal Camptothecins andUses Thereof,” is hereby incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

This invention relates to liposomal compositions and methods of usingsuch compositions for treating neoplasia and for inhibitingangiogenesis.

Many anticancer or antineoplastic drugs have been encapsulated inliposomes. These include alkylating agents, nitrosouras, cisplatin,antimetabolites, and anthracyclines. Studies with liposomes containinganthracycline antibiotics have clearly shown reduction of cardiotoxicityand dermal toxicity and prolonged survival of tumor bearing animalscompared to controls receiving free drug.

Liposomal anticancer drugs modify drug pharmacokinetics as compared totheir free drug counterpart. For a liposomal drug formulation, drugpharmacokinetics will be largely determined by the rate at which thecarrier is cleared from the blood and the rate at which the drug isreleased from the carrier. Considerable efforts have been made toidentify liposomal carrier compositions that show slow clearance fromthe blood and long-circulating carriers have been described in numerousscientific publications and patents. Efforts have also been made tocontrol drug leakage rates from liposomal carriers, using for example,transmembrane potential to control release.

Therapeutic camptothecins, such as Topotecan(9-dimethylaminomethyl-10-hydroxy-camptothecin; Hycamtin™), andIrinotecan, are a semi-synthetic, water soluble derivative ofcamptothecin, an alkaloid extracted from the stem wood of the Chinesetree Camptotheca acuminata (Wall, et al., J. Am. Chem. Soc. 88:3888-3890(1966)). Camptothecins belong to the topoisomerase inhibitor class ofantineoplastic agents, specifically inhibiting the action of the nuclearenzyme topoisomerase I which is involved in DNA replication (Hsiang, etal., Cancer Res. 48:1722-1726 (1988)). As such, topotecan exhibits acell cycle-specific mechanism of action, acting during S-phase (DNAreplication) to cause irreversible double strand breaks in DNA thatultimately lead to G2 cell cycle arrest and apoptosis. In the free form,the drug has a broad spectrum of activity against a range of tumor celllines and murine allograft and human xenograft tumor models (McCabe, F.L. et al., Cancer Invest 12:308-313 (1994); Emerson, et al., Cancer Res.55:603-609 (1995); Thompson, Biochim. Biophys. Acta 1400:301-319 (1998);Ormrod, et al., Drugs 58:533-551 (1999); Hardman, et al., AnticancerRes. 19:2269-2274 (1999)). More recently, evidence has emerged thattopotecan has strong anti-angiogenic properties that may contribute toits anti-tumor mechanism of action (O'Leary, et al., Clin. Cancer Res.5:181-187 (1999); Clements, et al., Cancer Chemother. Pharmacol.44:411-416 (1999)). All these treatments are associated withdose-limiting toxicity such as non-cumulative myelosuppression leadingto anaemia, neutropenia and thrombocytopenia, andgastrointestinal-related toxicity, including mucositis and diarrhea.Clinically, topotecan has been approved for second-line therapy inovarian and small cell lung cancer (SCLC) and is currently the focus ofextensive clinical evaluation.

Lipid formulations of camptothecins have been proposed as therapeuticagents (see, U.S. Pat. No. 5,552,156 and PCT Publication No. WO95/08986. However, not all lipid formulations are equal for drugdelivery purposes and extensive research continues into formulationswhich demonstrate preferred characteristics for drug loading andstorage, drug administration, pharmacokinetics, biodistribution, leakagerates, tumor accumulation, toxicity profile, and the like. Withcamptothecins, the field is further complicated because dose limitingtoxicities in humans may be 10-fold lower than in mice (Erickson-Miller,et al., Cancer Chemother. Pharmacol. 39:467-472 (1997)).

Improved liposomal formulations of antineoplastic agents could provevery useful. It is an object of the instant invention to provide lipidformulated antineoplastic agents having novel clinical utility.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful formodulating the plasma circulation half-life of an active agent (e.g.,topotecan). The liposomal formulations have increased clinical efficacyand decreased collateral toxicity. In addition, the present inventionprovides methods and liposomal compositions for treating neoplasia andinhibiting angiogenesis.

As such, in one embodiment, the present invention provides a method formodulating the plasma circulation half-life of an active agent,comprising: (a) providing a liposome having free active agent andprecipitated active agent encapsulated therein; and (b) varying theamount of the active agent that is precipitated in the liposome.Surprisingly, by varying the amount of active agent that is precipitatedin the liposome, it is possible to modulate the release kinetics of theactive agent into the plasma. Preferred active agents are antineoplasticdrugs, such as a camptothecin (e.g., topotecan).

In another embodiment, the present invention provides a liposomalformulation, comprising: a) an antineoplastic drug; and b) a liposomehaving free antineoplastic drug and precipitated antineoplastic drug,wherein the precipitated antineoplastic drug in the liposome is at least50% of the total antineoplastic drug. By tailoring the amount ofprecipitated antineoplastic drug in the liposome, it is possible tocontrol the release of the drug, both in vitro and in vivo. In certainpreferred embodiments, high intraliposomal concentrations of the activeagent (e.g., topotecan) results in a high amount of precipitated form.In this aspect, subsequent release rates of the drug in vivo are slow.In certain aspects, a slow release rate is preferable and moreefficacious compared to a fast release rate.

In yet another embodiment, the present invention provides a liposomalformulation, comprising: a) an active agent; b) a liposome having freeactive agent and precipitated active agent encapsulated therein; and c)an empty liposome.

In this aspect, the serum half-life of the liposome is prolonged byincluding empty liposomes in the formulation. It will be readilyapparent to those of skill in the art that any of a variety of lipidscan be used to form the liposomal compositions of the present invention.In a presently preferred embodiment, the lipid comprises a mixture ofsphingomyelin and cholesterol, preferably at a spingomyelin:cholesterolratio (molar ratio) of about 30:70 to about 60:40. In one preferredembodiment, the liposome comprises sphingomyelin and cholesterol in a55:45 ratio.

In still another aspect, the present invention provides a method oftreating a solid tumor in a human afflicted therewith, the methodcomprising administering to the human an effective amount of a liposomalformulation of the present invention in a pharmaceutically acceptablecarrier. A variety of solid tumors can be treated using the compositionsof the present invention. In a preferred embodiment, the solid tumor tobe treated is selected from the group consisting of solid tumors of thelung, mammary, colon and prostate. In another preferred embodiment, themethod further comprises co-administration of a treatment or activeagent suitable for treating neutropenia or platelet deficiency.

In a preferred embodiment, a liposomal topotecan is used to treat thesolid tumors. In addition, it will be readily apparent to those of skillin the art that any of a variety of lipids can be used to form theliposomal compositions of the present invention.

Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detailed descriptionwhich follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C shows the pharmacokinetic behavior of a liposomal formulationof vinorelbine. Panel A shows the rates of drug leakage from circulatingcarriers for three formulations of differing drug:lipid ratio (0.1:1,0.2:1, 0.3:1). Drug release is dependent upon drug:lipid ratio with theslowest rate of release seen for the highest ratio (0.3:1). Panel Bshows lipid recovery in the blood. Panel C shows that modulation in drugrelease rates from the carrier results in changes to the blood clearancehalf-life for vinorelbine.

FIG. 2A-C shows a corresponding behavior when plasma drug levels areused to follow pharmacokinetics. Panel A shows drug retention versustime. Panel B shows lipid recovery versus time. Panel C shows drugrecovery versus time.

FIG. 3A-C shows the pharmacokinetic behavior of formulations ofliposomal vinblastine as a function of drug:lipid ratio (blood PK). Drugleakage from the liposomal carrier is determined by the initialdrug:lipid ratio with slower release for formulations of higher drugratio. Panel A shows drug retention versus time. Panel B shows lipidrecovery versus time. Panel C shows drug release rates correlate withchanges to drug clearance half-life from the blood.

FIG. 4A-C shows the pharmacokinetic behavior of formulations ofliposomal vinblastine as a function of drug:lipid ratio (plasma PK).Panel A shows drug retention versus time. Panel B shows lipid recoveryversus time. Panel C shows drug release rates correlate with changes todrug clearance half-life from the plasma.

FIG. 5A-C shows the influence of lipid dose on PK behavior (blood PK).As illustrated therein, similar rates of drug release (A), lipidclearance (B) and drug clearance (C) are seen for a liposomalvinblastine formulation of drug:lipid ratio 0.3:1 over a lipid doserange of 16.6 mg/kg to 50 mg/kg.

FIG. 6A-C shows the influence of lipid dose on PK behavior (plasma PK).As illustrated therein, similar rates of drug release (A), lipidclearance (B) and drug clearance (C) are seen for a liposomalvinblastine formulation of drug:lipid ratio 0.3:1 over a lipid doserange of 16.6 mg/kg to 50 mg/kg.

FIG. 7A-B shows the pharmacokinetic behavior of two formulations ofliposomal topotecan of differing drug:lipid ratios. Panel A shows thatwhen topotecan is loaded to a drug:lipid ratio of 0.11:1, a much slowerdrug release rate is seen resulting in a much longer plasma clearancerate compared to Panel B having a formulation of lower drug:lipid ratioof 0.02:1.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The activity of many anticancer drugs is dependent on theirpharmacokinetic behavior. This pharmacokinetic behavior defines the drugconcentrations and period of time over which cancer cells are exposed tothe drug. In the case of most anticancer drugs, longer exposure timesare preferred as this results in increased killing of the cancer cells.In general, several parameters are used to describe drugpharmacokinetics. Plasma clearance half-time and area under the curve(AUC) are examples. The plasma clearance half-time is the time requiredfor half of the administered drug to be removed from the plasma. The AUCis a measure of plasma drug levels over time and provides an indicationof the total drug exposure. Generally, increased plasma clearancehalf-life and plasma AUC for an anticancer drug correlate with increasedtherapeutic efficacy.

I. Modulating Active Agent Release

The present invention describes methods and formulations for modulatingdrug release from liposomes. In one embodiment, the present inventionprovides a method for modulating the plasma circulation half-life of anactive agent, comprising: (a) providing a liposome having free activeagent and precipitated active agent encapsulated therein; and (b)varying the amount of the active agent that is precipitated in theliposome. Preferably, the “free active agent” and the “precipitateactive agent” are the same active agent, however the present inventionis not so limited. As used herein, the term “modulating” can mean eitherincreasing or decreasing the release rate of the active agent from theliposomal carrier. For antineoplastic active agents, modulating ispreferably decreasing or slowing the release rate of the active agent.

In preferred aspects, the liposomes of the present invention containboth encapsulated free active agent and precipitated active agent. Theamount of active agent that is precipitated within the liposome can bevaried using a variety of mechanisms. For example, by varying the activeagent to lipid ratio the amount of active agent that is precipitated canbe increased or decreased. Drug loading at low drug:lipid ratios,results in low concentrations of active agent (e.g., topotecan) in theliposome interior and hence most, if not all of the entire drug is insolution i.e., not precipitated or free. Low precipitation amountsresult in a fast release rate of the drug from the liposome. Conversely,a high drug:lipid ratio results in high intraliposomal concentrationsand high precipitation amounts. When the drug is in a precipitated form,subsequent release rates in vivo or in vitro are slow. Forantineoplastic drugs (e.g., topotecan), slow release rates arepreferable.

Without being bound by any particular theory, it is believed that theliposomes of the present invention undergo a “precipitation-dissolutionmechanism” (PDM), which dictates drug release. In the PDM mechanism ofthe present invention, the dissolution rate of precipitated active agent(e.g., topotecan) within the lipsomome's interior into the internalsolution of the liposome is slow, compared to the rate of release ofactive agent out of the liposome to the exterior and is thus ratedetermining. That is, the rate of dissolution of the precipitated drugto free drug in the liposome's interior determines how fast the drugwill be released into the plasma.

In certain embodiments, the active agent to lipid ratio can be varied bythe addition of empty liposomes. In general, liposomes whether empty orthose having active agents contained therein are cleared by cells of thereticuloendothelial system (RES). Typically, the RES will remove 80-95%of a dose of injected liposomes within one hour, effectivelyout-competing the selected target site for uptake of the liposomes. Avariety of factors which influence the rate of RES uptake of liposomeshave been reported including, liposome size, charge, degree of lipidsaturation, and surface moieties. By including empty liposome vesicles,it is possible to shield the liposomes containing active agent from theRES. Thus, empty liposome vesicles actually extend the blood circulationlifetime of the liposomes by acting as “decoys”. An extended circulationtime is often needed for liposomes to reach the target region, cell orsite from the site of injection. The empty liposomal vesicles keep theRES busy and as a result, the serum half-life of the liposomes havingactive agent contained therein is increased.

In certain other aspects, a component(s) is added to the liposome thatwill enhance the precipitation of the active agent. In this aspect, avariety of charged ions can be used to increase the amount ofprecipitated active agent in the vesicle's interior. In preferredaspects, divalent, trivalent or polyvalent anions are used. Suitableanions include, but are not limited to, carboxylate (—CO₂ ⁻), sulfonate(SO₃ ⁻), sulfate (SO₄ ⁻²), hydroxide (—OH), alkoxides, phosphate (—PO₄⁻²), and phosphonate (—PO₃ ⁻²). Those of skill in the art will know ofother components, which will enhance the amount of precipitated activeagent in the liposome's interior.

Moreover, the drug:lipid ratios can be varied using the size of theliposome. The larger the liposome vesicle used, the smaller thedrug:lipid ratio. In certain aspects, both the active agent to lipidratio and the size of the liposome are varied to optimize the efficacyof the active agent.

The amount of encapsulated active agent that is precipitated in vesiclewill vary and is somewhat dependent on the active agent itself. Incertain embodiments, the amount of precipitated active agent is at leastabout 25% to about 95% (such as about 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%) of total active agent. Fortopotecan, the amount of the precipitated active agent encapsulated inthe liposome is at least 50% of the total active agent.

In preferred aspects, when the active agent is an antineoplastic drug,using higher drug:lipid ratios results in higher amounts of encapsulatedprecipitated drug. As a result, drug release from the liposomes in vivois slower than for similar compositions prepared at lower drug:lipidratio. These higher drug:lipid ratio liposomes exhibit extended plasmahalf-life and increased plasma AUC values. Advantageously, theseformulations exhibit improved antitumor efficacy.

In certain embodiments, the ratio of active agent:lipid is about0.005-1:1 (w/w).

Preferably, the ratio of active agent:lipid is about 0.05-0.9:1 (w/w)and more preferably, the ratio of active agent:lipid is about 0.1-0.5:1(w/w). By modulating the plasma circulation half-life of the activeagent, it is thus possible to maximize or optimize efficacy of theactive agent.

II. Compositions and Methods of Making Liposomal Formulations

Liposome, vesicle and liposome vesicle will be understood to indicatestructures having lipid-containing membranes enclosing an aqueousinterior. The structures can have one or more lipid membranes unlessotherwise indicated, although generally the liposomes will have only onemembrane. Such single-layered liposomes are referred to herein as“unilamellar.” Multilayer liposomes are referred to herein as“multilamellar.”

The liposomes that are used in the present invention are preferablyformed from lipids which when combined form relatively stable vesicles.An enormous variety of lipids are known in the art, which can be used togenerate such liposomes. Preferred lipids include, but are not limitedto, neutral and negatively charged phospholipids or sphingolipids andsterols, such as cholesterol. The selection of lipids is generallyguided by consideration of, e.g., liposome size and stability of theliposomes in the bloodstream.

Preferred liposome compositions for use in the present invention includethose comprising sphingomyelin and cholesterol. The ratio ofsphingomyelin to cholesterol in the liposome composition can vary, butgenerally is in the range of from about 75/25 mol %/mol %sphingomyelin/cholesterol to about 30/50 mol %/mol %sphingomyelin/cholesterol, more preferably about 70/30 mol %/mol %sphingomyelin/cholesterol to about 40/45 mol %/mol %sphingomyelin/cholesterol, and even more preferably about 55/45 mol%/mol % sphingomyelin/cholesterol. Other lipids can be included in theliposome compositions of the present invention as may be necessary, suchas to prevent lipid oxidation or to attach ligands onto the liposomesurface. Generally, if lipids are included, the other inclusion of suchlipids will result in a decrease in the sphingomyelin/cholesterol ratio.Liposomes of this type are known as sphingosomes and are more fullydescribed in U.S. Pat. No. 5,814,335, the teachings of which areincorporated herein by reference.

A variety of methods are available for preparing liposomes as describedin, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U.S.Pat. Nos. 4,235,871; 4,501,728; 4,837,028, the text Liposomes, Marc J.Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, etal, Chem. Phys. Lip. 40:89 (1986), all of which are incorporated hereinby reference. The protocol for generating liposomes generally includes:mixing of lipid components in an organic solvent; drying andreconstituting liposomes in aqueous solvent; and sizing of liposomes(such as by extrusion), all of which are well known in the art.

Alternative methods of preparing liposomes are also available. Forinstance, a method involving detergent dialysis based self-assembly oflipid particles is disclosed and claimed in U.S. Pat. No. 5,976,567issued to Wheeler, et al., which avoids the time-consuming and difficultto-scale drying and reconstitution steps. Further methods of preparingliposomes using continuous flow hydration are under development and canoften provide the most effective large scale manufacturing process.

Preparation of liposomal formulations having active agents (e.g.,camptothecins) requires loading of the drug into the liposomes. Loadingcan be either passive or active. Passive loading generally requiresaddition of the drug to the buffer at the time of the reconstitutionstep. This allows the drug to be trapped within the liposome interior,where it will remain if it is not lipid soluble, and if the vesicleremains intact (such methods are employed, for example, in PCTPublication No. WO 95/08986, the teachings of which are incorporatedherein by reference).

Active loading is in many ways preferable, and a wide variety oftherapeutic agents can be loaded into liposomes with encapsulationefficiencies approaching 100% by using a transmembrane pH or iongradient (see, Mayer, et al., Biochim. Biophys. Acta 1025:143-151 (1990)and Madden, et al., Chem. Phys. Lipids 53:37-46 (1990)). Numerous waysof active loading are known to those of skill in the art. All suchmethods involve the establishment of some form of gradient that drawslipophilic compounds into the interior of liposomes where they canreside for as long as the gradient is maintained. Very high quantitiesof the desired drug can be obtained in the interior, so much that thedrug may precipitate out on the interior and generate a continuinguptake gradient.

Particularly preferred for use with the instant invention isionophore-mediated loading as disclosed and claimed in U.S. Pat. No.5,837,282, the teachings of which are incorporated by reference herein.The ionophore-mediated loading is an electroneutral process and does notresult in formation of a transmembrane potential. With hydrogen iontransport into the vesicle there is concomitant magnesium ion transportout of the vesicle in a 2:1 ratio (i.e. no net charge transfer). In thecase of topotecan, it is thought that the agent crosses the membrane ina neutral state (no charge). Upon entry into the vesicle, topotecanbecomes positively charged. As ionophore-mediated loading is anelectroneutral process, there is no transmembrane potential generated.

An important characteristic of liposomal camptothecins forpharmaceutical purposes is the drug to lipid ratio of the finalformulation. As discussed earlier, drug:lipid ratios can be establishedin two ways: 1) using homogenous liposomes each containing the samedrug:lipid ratio; or 2) by mixing empty liposomes with liposomes havinga high drug:lipid ratio to provide a suitable average drug:lipid ratio.For different applications, different drug:lipid ratios may be desired.Techniques for generating specific drug:lipid ratios are well known inthe art. Drug:lipid ratios can be measured on a weight to weight basis,a mole to mole basis or any other designated basis. Preferred drug:lipidratios range from about 0.005:1 drug:lipid (by weight) to about 0.2:1drug:lipid (by weight) and, more preferably, from about 0.1:1 drug:lipid(by weight) to about 0.3:1 drug:lipid (by weight).

A further important characteristic is the size of the liposomeparticles. For use in the present inventions, liposomes having a size offrom about 0.05 microns to about 0.15 microns are preferred.

The present invention also provides liposomal compositions (e.g.,camptothecin) in kit form. The kit can comprise a ready-madeformulation, or a formulation, which requires mixing of the medicamentbefore administration. The kit will typically comprise a container thatis compartmentalized for holding the various elements of the kit. Thekit will contain the liposomal compositions of the present invention orthe components thereof, possibly in dehydrated form, with instructionsfor their rehydration and administration

The liposome compositions prepared, for example, by the methodsdescribed herein can be administered either alone or in a mixture with aphysiologically acceptable carrier (such as physiological saline orphosphate buffer) selected in accordance with the route ofadministration and standard pharmaceutical practice. Generally, normalsaline will be employed as the pharmaceutically acceptable carrier.Other suitable carriers include, e.g., water, buffered water, 0.4%saline, 0.3% glycine, and the like, including glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions may also contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.Additionally, the composition may include lipid-protective agents, whichprotect lipids against free-radical and lipid-peroxidative damages onstorage. Lipophilic free-radical quenchers, such as α.-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

A wide variety of active agents are suitable for the liposomalcompositions and methods of the present invention. In a preferredaspect, the active agents are antineoplastic drugs. Currently, there areapproximately twenty recognized classes of approved antineoplasticdrugs. The classifications are generalizations based on either a commonstructure shared by particular drugs, or are based on a common mechanismof action by the drugs. A partial listing of some of the commonly knowncommercially approved (or in active development) antineoplastic agentsby classification is as follows:

Structure-Based Classes:

1. Fluoropyrimidines—5-FU, Fluorodeoxyuridine, Ftorafur,5′-deoxyfluorouridine, UFT, S-1 Capecitabine;

2. Pyrimidine Nucleosides—Deoxycytidine, Cytosine Arabinoside,5-Azacytosine, Gemcitabine, 5-Azacytosine-Arabinoside;

3. Purines—6-Mercaptopurine, Thioguanine, Azathioprine, Allopurinol,Cladribine, Fludarabine, Pentostatin, 2-Chloro Adenosine;

4. Platinum Analogues—Cisplatin, Carboplatin, Oxaliplatin, Tetraplatin,Platinum-DACH, Ormaplatin, CI-973, JM-216;

5. Anthracyclines/Anthracenediones—Doxorubicin, Daunorubicin,Epirubicin, Idarubicin, Mitoxantrone;

6. Epipodophyllotoxins—Etoposide, Teniposide;

7. Camptothecins—Irinotecan, Topotecan, 9-Amino Camptothecin,10,11-Methylenedioxy Camptothecin, 9-Nitro Camptothecin, TAS 103,7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin,7-(2-N-isopropylamino)ethyl)-20(S)-camptothecin;

8. Hormones and Hormonal Analogues—Diethylstilbestrol, Tamoxifen,Toremefine, Tolmudex, Thymitaq, Flutamide, Bicalutamide, Finasteride,Estradiol, Trioxifene, Droloxifene, Medroxyprogesterone Acetate,Megesterol Acetate, Aminoglutethimide, Testolactone and others;

9. Enzymes, Proteins and Antibodies—Asparaginase, Interleukins,Interferons, Leuprolide, Pegaspargase, and others;

10. Vinca Alkaloids—Vincristine, Vinblastine, Vinorelbine, Vindesine;

11. Taxanes—Paclitaxel, Docetaxel.

Mechanism-Based Classes:

1. Antihormonals—See classification for Hormones and Hormonal Analogues,Anastrozole;

2. Antifolates—Methotrexate, Aminopterin, Trimetrexate, Trimethoprim,Pyritrexim, Pyrimethamine, Edatrexate, MDAM;

3. Antimicrotubule Agents—Taxanes and Vinca Alkaloids;

4. Alkylating Agents (Classical and Non-Classical)—Nitrogen Mustards(Mechlorethamine, Chlorambucil, Melphalan, Uracil Mustard),Oxazaphosphorines (Ifosfamide, Cyclophosphamide, Perfosfamide,Trophosphamide), Alkylsulfonates (Busulfan), Nitrosoureas (Carmustine,Lomustine, Streptozocin), Thiotepa, Dacarbazine and others;

5. Antimetabolites—Purines, pyrimidines and nucleosides, listed above;

6. Antibiotics—Anthracyclines/Anthracenediones, Bleomycin, Dactinomycin,Mitomycin, Plicamycin, Pentostatin, Streptozocin;

7. Topoisomerase Inhibitors—Camptothecins (Topo I), Epipodophyllotoxins,m-AMSA, Ellipticines (Topo II);

8. Antivirals—AZT, Zalcitabine, Gemcitabine, Didanosine, and others;

9. Miscellaneous Cytotoxic Agents—Hydroxyurea, Mitotane, Fusion Toxins,PZA, Bryostatin, Retinoids, Butyric Acid and derivatives, Pentosan,Fumagillin, and others.

The objective of all antineoplastic drugs is to eliminate (cure) or toretard the growth and spread (remission) of the cancer cells. Themajority of the above listed antineoplastic agents pursue this objectiveby possessing primary cytotoxic activity, effecting a direct kill on thecancer cells. Other antineoplastic drugs stimulate the body's naturalimmunity to effect cancer cell kill. The literature is replete withdiscussions on the activity and mechanisms of all of the above drugs,and many others.

Exemplary methods of making specific formulations of liposomalcamptothecins and, in particular, liposomal topotecan are set out in theexamples below.

III. Methods of Using Liposomal Camptothecins

The liposomal compositions (e.g., camptothecins) of the presentinvention are used, in the treatment of solid tumors in an animal, suchas a human. The examples below set out key parameters of the drug:lipidratios, dosages of active agent and lipid to be administered, andpreferred dose scheduling to treat different tumor types.

Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously or intramuscularly. More preferably, the pharmaceuticalcompositions are administered by intravenous drip or intraperitoneallyby a bolus injection. The concentration of liposomes in thepharmaceutical formulations can vary widely, i.e., from less than about0.05%, usually at or at least about 2-5% to as much as 10 to 30% byweight and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.For example, the concentration can be increased to lower the fluid loadassociated with treatment. Alternatively, liposomes composed ofirritating lipids can be diluted to low concentrations to lesseninflammation at the site of administration. The amount of liposomesadministered will depend upon the particular camptothecin used, thedisease state being treated and the judgement of the clinician, but willgenerally, in a human, be between about 0.01 and about 50 mg perkilogram of body weight, preferably between about 5 and about 40 mg/kgof body weight. Higher lipid doses are suitable for mice, for example,50-120 mg/kg.

Dosage for the active agent (e.g., camptothecin) will depend on theadministrating physician's opinion based on age, weight, and conditionof the patient, and the treatment schedule. A recommended dose for freetopotecan in Small Cell Lung Cancer is 1.5 mg/M² per dose, every day for5 days, repeated every three weeks. Because of the improvements intreatment now demonstrated in the examples, below, doses of active agent(e.g., topotecan) in humans will be effective at ranges as low as from0.015 mg/M²/dose and will still be tolerable at doses as high as 15 to75 mg/M²/dose, depending on dose scheduling. Doses may be single dosesor they may be administered repeatedly every 4 h, 6 h, or 12 h or every1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 9 d, 10 d or combinationthereof. Preferred scheduling may employ a cycle of treatment that isrepeated every week, 2 weeks, three weeks, four weeks, five weeks or sixweeks or combination thereof. In a presently preferred embodiment,treatment is given once a week, with the dose typically being less than1.5 mg/M².

Particularly preferred topotecan dosages and scheduling are as follows:

Dosage (mg/M²/dose) Period Repeat Cycle every: 0.15 1 d × 5 d 3 weeks0.5 1 d 1 week 1.5 1 d 1 week 15 1 d 3 weeks 50 1 d 3 weeks

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters, which can be changed or modified to yield essentially thesame results.

IV. EXAMPLES

A. Materials and Methods

1. Materials. Topotecan (Hycamtin™, SmithKline Beecham) was purchasedfrom the pharmacy at the British Columbia Cancer Agency. Sphingomyelin(SM) was purchased from Avanti Polar Lipids. Sphingomyelin from NorthernLipids was used in an early study, but was less soluble in ethanol thanthe Avanti version. Cholesterol (CH) and the divalent cation ionophoreA23187 were purchased from Sigma. [³H]-cholesterylhexadecylether(Dupont) was used as a lipid marker.

2. Mice. Female, ICR, BDF-1 or athymic nu/nu (6-8 weeks) were purchasedfrom Harlan-Sprague Dawley (Indianapolis, Ind.). All animals werequarantined for one week prior to use. All studies were conducted inaccordance with the guidelines established by the Canadian Council onAnimal Care (CCAC) and the Institutional Animal Care and User Committee(IACUC).

3. Formulation of topotecan by the Mg-A23187 method. Topotecan wasencapsulated in SM:CH (55:45, mol/mol) liposomes using the Mg-A23187ionophore method according to U.S. Pat. No. 5,837,282. The initialdrug-to-lipid ratio was 0.10 (w/w) and drug loading was typically95-100%. The external buffer consisted of 10 mM PBS, pH 7.5 and 300 mMsucrose. All formulations were analyzed with respect to particle size,drug loading efficiency, pH, and drug and lipid concentration.

4. Drug preparation and dosing. Each vial of topotecan (Hycamtin™) washydrated in 1.0 ml of sterile water, giving a topotecan concentration of4.0 mg/ml. Subsequent dilutions were μl made in 0.9% sterile saline tomaintain the low pH required for the lactone species of the drug. Unuseddrug in the water stock solution (4.0 mg/ml) was stored at 4° C. in theabsence of light. Liposome encapsulated topotecan was diluted in 0.9%saline to the required concentration for administration. All drugadministrations were at 10 ml/kg (200 μl/20 g mouse) via the lateraltail vein.

5. Pharmacokinetic and in vivo leakage studies. The pharmacokinetics anddrug leakage of free and liposome encapsulated topotecan were evaluatedin ICR mice over 24 h following i.v. administration via the lateral tailvein. Two different drug-to-lipid ratios, i.e., 0.10 (w/w) and 0.02(w/w), were used to examine the influence of drug-to-lipid ratio andlipid dose on drug leakage and PK behavior. Encapsulated topotecan wasadministered at 1 mg/kg (10 or 50 mg/kg lipid) and 5 mg/kg topotecan (50mg/kg lipid). Correspondingly, the PK behavior of free topotecan wasevaluated at and 1 and 5 mg/kg. Total topotecan in blood was determinedby a fluorescence assay preceded by precipitation of plasma proteins.Topotecan was quantified by spectrofluorimetry at an excitation (2.5 nmslit width) and emission wavelength (2.5 nm slit width) of 380 and 518nm, respectively. Lipid levels in plasma were determined by liquidscintillation counting of the [³H]-CHE label.

6. MTD studies. MTD studies were performed in the host mouse straincorresponding to each tumor model. Single dose and multidose MTD weredetermined by monitoring weight loss over time. The MTD was defined asthe dose that resulted in 20% weight loss.

7. Myelosuppression and neutropenia studies. Alteration in peripheralblood cell levels as a consequence of topotecan administration wasassessed over 4-6 weeks in ICR mice. Blood was collected into EDTAmicrotainer tubes at Day 1, 3, 5, 7, 14, and 21 following i.v.administration of free or liposome encapsulated topotecan at 10 mg/kg.Empty vesicles were administered as a control. CBC and differentialanalysis was performed at Central Labs for Veterinarians (Langley, BC)to quantify cellular levels, ratios and morphology.

8. Tumor Models. The L1210 murine leukemia model and the CT-26 murinecolon metastases model were employed as in standard protocols. HumanMX-1 and LX-1 cell lines were obtained from the DCTD Tumor Repository inFrederick, Md. These cell lines were received as tumor fragments andwere propagated in NCr nude mice by serial transplantation of 3×3 mmfragments. Experiments were not initiated until the cell lines had beenthrough 3 passages in nude mice and the tumor lines were restarted whenthe passage number reached 10.

9. Efficacy Studies. All dosing of free and liposomal topotecan wasadministered by the intravenous route at 10 ml/kg via the lateral tailvein. In the L1210 and CT-26 models, dosing occurred on day 1 (tumorcell injection=day 0). For the MX-1 and LX-1 tumor models, tumor volumewas determined by repeated perpendicular measurements of tumordimensions and using the formula:Volume (mm³)=(L×W ²)/2

Dosing was initiated in the MX-1 and LX-1 models when tumors had clearlydemonstrated growth and were in the range 100-300 mm³.

Since most drugs exhibit a balance between a biological effect andtoxicity, it is useful to examine a parameter that incorporates both ofthese attributes. The most commonly employed parameter is therapeuticindex (TI). Traditionally, therapeutic index is defined as:TI=LD ₅₀ /ED ₅₀

However, since it is no longer permissible to perform LD50 studies,therapeutic index for these studies has been defined as follows:TI=MTD/MED.

In the above formula, MTD is the maximum tolerated dose, defined as thatdose that causes a mean weight loss of 20% in a group of animals; andMED is the minimal effective dose, defined as the dose that produces anoptimal % T/C value of ≦40 in the solid tumor models or an % ILS of50±10% in the survival models.

B. Results

1. Pharmacokinetics and drug leakage. The influence of liposomeencapsulation and drug-to-lipid ratio on plasma pharmacokinetics anddrug leakage of topotecan was examined over 24 h in ICR mice. Liposomeencapsulation of topotecan (drug-to-lipid ratio, 0.11, wt/wt) had adramatic influence on the pharmacokinetics parameters of the drug (see,FIG. 1, top; and Table 1). At a 5 mg/kg dose of topotecan, a 164-foldincrease in plasma AUC, a 24-fold increase in C_(max) and a 24-foldincrease in the plasma α half-life were observed for the liposomal drugrelative to the free drug (see, Table 1). Historically, largeimprovements in AUC and plasma half-lives of liposomal drugs haveresulted in enhanced delivery of the drug to disease-sites (such astumors), a process known as “disease-site targeting”.

The formulations used in this study were prepared by the Mg-A23187ionophore method. There was an initial rapid release of drug in thefirst 10-30 minutes after iv administration (see, FIG. 1, bottom),followed by a more gradual release phase. The t,₁₂release for theMn-A23187 and Mg-A23187 formulations were ˜3 h and ˜5-7 h, respectively;however, very little drug was present in either formulation at 24 h.

For most liposomal drug formulations, the pharmacokinetic properties ofthe encapsulated drug are controlled by the lipid composition and dose.Liposomal topotecan has been shown to exhibit exceptional anti-tumoractivity, even at very low drug doses (0.5 mg/kg; drug-to-lipid ratio,0.10, wt/wt). At these drug doses and drug-to-lipid ratio, liposomeelimination from the plasma is expected to be rapid. Therefore, todetermine whether the pharmacokinetics of topotecan at low doses couldbe improved, a low drug-to-lipid ratio (0.02, wt/wt) formulation oftopotecan was investigated. Interestingly, in this study, the lowdrug-to-lipid ratio formulation released the drug much faster than thehigher drug-to-lipid ratio (0.11, wt/wt) formulation. This result wasunexpected.

TABLE 1 Pharmacokinetic parameters of free and liposomal topotecan. DoseAUC Cmax Cl α_(½) β_(½) Formulation (mg/kg) (h · μg/ml) (μg/ml) (ml/h)(h) (h) Free 1 1.97 0.75 13.9 0.14 11.8 5 2.77 2.17 49.6 0.26 11.4 TCS 165.7 16.3 0.417 2.79 5 453 51.0 0.302 6.16All parameters were derived from one or two-compartment models usingWINNONLIN PK modeling software.

2. Maximum tolerated doses. Single and multidose MTD studies wereperformed in tumor bearing Balb/c, BDF-1 and NCr nu/nu mice. Bodyweights of individual mice were monitored throughout each study toevaluate the general tolerability of free and liposomal topotecan and,where possible, to establish an MTD (see, FIG. 2). The maximum tolerateddose of liposomal topotecan was 10 mg/kg on a single administration, 7.5mg/kg on a q7dx3 schedule and 5 mg/kg on a q3dx4 schedule. The reportedLD₁₀ of free topotecan following a single intravenous infusion in miceis 75 mg/M² (˜25 mg/kg) [Hycamtin™ product monograph]; however, verylittle weight loss was observed at doses up to 40 mg/kg, although thiswas considered the MTD due to acute responses. Drug quantities werelimited so doses higher than 40 mg/kg (administered over 5-10 minutes)were not pursued. It has previously been indicated that the LD₁₀ of freetopotecan on a qdx5 schedule is 14 mg/M2/dose (˜4.7 mg/kg/dose)(Grochow, , et al., Drug Metab. Dispos. 20:706-713 (1992)).

3. Toxicity. The major dose-limiting toxicity of free topotecanadministered daily in humans for 5 consecutive days (dx5) at 1.5 mgM²/dose, the MTD, is non-cumulative myelosuppression. As mentionedearlier, humans are more sensitive than mice to myelosuppression and canonly tolerate 11% of the MTD in mice (1.5 vs 14 mg/M²). In this regard,dogs have been shown to be a much better predictor of topotecanmyelosuppression in humans (Burris, et al., J. Natl. Cancer Inst.84:1816-1820 (1992)). However, mice should be suitable for comparing therelative myelosuppressive effects of free and liposome encapsulatedtopotecan.

In a study, the maximal reduction in peripheral WBC counts occurred atday 3 post-injection following administration of liposomal topotecan. Acomparison of peripheral blood cell levels and morphology was then madeat day 3 following administration of free or liposome encapsulatedtopotecan or empty vesicles (see, Table 2). The dose used for thiscomparison was the MTD of liposome-encapsulated topotecan (10 mg/kg). Asignificant reduction in circulating neutrophils was observed forliposomal topotecan relative to free topotecan (˜10-fold), emptyvesicles (˜10-fold) or control animals (˜20-fold). Total WBC levels andthe lymphocyte sub-population were reduced approximately 2-fold forliposomal topotecan relative to control animals. No significantdifferences were observed in these parameters for free topotecan at thesame dose. At day 21 post-injection total, WBC levels for liposomaltopotecan remained approximately 2.5-fold lower than normal animals;however, neutrophils levels had recovered from a 20-fold decrease to a3-fold decrease relative to normal mice. Lymphocyte levels remained˜2-fold lower than normal mice. No other significant differences wereobserved.

Analysis of serum chemistry parameters at day 3 post-injection revealedvery few changes relative to untreated animals (see, Table 3). The onlychange of note was a statistically significant increase (˜2-fold) inglobulin levels and a concomitant decrease in the albumin/globulin ratiofor animals treated with liposomal topotecan. No other significantchanges were observed.

TABLE 2 Blood CBC and differential of ICR mice treated with a 10 mg/kgi.v. dose of free or liposome encapsulated topotecan. Day WBCDifferential Post- WBC Neutro Lympho Mono Eosino Baso TreatmentInjection (×10⁹/L) (×10⁹/L) (×10⁹/L) (×10⁹/L) (×10⁹/L) (×10⁹/L) Control6.47 ± 1.62 0.937 ± 0.201 5.23 ± 1.45 0.180 ± 0.042 0.059 ± 0.039 0.056± 0.053 Free 3 6.70 ± 1.95 0.520 ± 0.200 5.90 ± 1.70 0.177 ± 0.072 0.031± 0.021 0.057 ± 0.040 21 5.16 ± 1.18 0.480 ± 0.122 4.33 ± 0.93 0.247 ±0.180 0.034 ± .016  0.088 ± 0.071 TCS 3 2.82 ± 1.05 0.048 ± 0.018 2.63 ±0.87 0.109 ± 0.126 0.001 ± 0.001 0.034 ± 0.029 21 2.54 ± 1.43 0.282 ±0.167 2.06 ± 1.36 0.133 ± 0.142 0.019 ± 0.011 0.064 ± 0.060 Empty 3 4.68± 1.13 0.598 ± 0.238 3.66 ± 0.93 0.248 ± 0.168 0.081 ± 0.044 0.064 ±0.055 21 5.05 ± 0.64 0.898 ± 0.575 3.78 ± 0.88 0.263 ± 0.163 0.038 ±0.036 0.072 ± 0.057 RBC Hb Hc PLT Treatment (×10¹²/L) (g/L) (L/L)(×10⁹/L) Control 8.67 ± 0.93 142 ± 12 0.438 ± 0.045  717 ± 317 Free 8.47± 0.39 136 ± 05 0.444 ± 0.012  879 ± 145 9.81 ± 0.37 154 ± 04 0.493 ±0.014  907 ± 059 TCS 8.93 ± 0.76 141 ± 10 0.463 ± 0.033  564 ± 098 9.41± 0.83 154 ± 12 0.486 ± 0.035 1009 ± 161 Empty 7.77 ± 0.30 130 ± 050.416 ± 0.014  863 ± 143 9.36 ± 0.67 152 ± 08 0.483 ± 0.033 1366 ± 144

TABLE 3 Serum chemistry panel of ICR mice treated with a 10 mg/kg i.v.dose of free or liposome encapuslated topotecan-day 3 post-injection.BUN Creatinine TP Albumin Globulin Alb/Glob Bilirubin Alk Phos ALT ASTCPK Treatment (mmol/L) (μmol/L) (g/L) (g/L) (g/L) Ratio (μmol/L) (IU/L)(IU/L) (IU/L) (IU/L) Control 11.3 ± 3.0 83 ± 6  46.7 ± 2.1  31.3 ± 1.515.3 ± 1.2  2.07 ± 0.15 4.7 ± 0.6 86 ± 12 27 ± 31 59 ± 22  87 ± 107 Free 9.4 ± 3.2 82 ± 18 48.0 ± 2.1  32.8 ± 1.3 15.2 ± 1.1  2.16 ± 0.15 3.8 ±0.8 67 ± 35 13 ± 23 55 ± 10  56 ± 38  TCS 10.0 ± 3.9 96 ± 28 55.8 ± 11.828.8 ± 2.5 27.0 ± 10.1 1.18 ± 0.33 2.5 ± 0.6 73 ± 21 23 ± 17 77 ± 29 155± 54  Empty ND 68 ± 13 49.3 ± 1.2  33.0 ± 1.7 16.3 ± 0.6  2.00 ± 0.174.3 ± 0.6 70 ± 10 17 ± 15 53 ± 6   56 ± 26 

C. Efficacy Studies in Murine and Human Tumor Models: Single DoseStudies

1. L1210 Murine Leukemia. The intravenous L1210 murine leukemia modelhas been used extensively to evaluate differential activity between freeand liposome encapsulated chemotherapeutic agents and was one of theoriginal (1955-1975) models in the in vivo NCI screen of novelchemotherapeutic agents (Plowman, et al., Human tumor xenograft modelsin NCI drug development. In “Anticancer Drug Development Guide:Preclinical Screening, Clinical Trials, and Approval” (B. Teicher, Ed.),Humana Press Inc., Totowa (1997); Waud, Murine L1210 and P388 leukemias.In “Anticancer Drug Development Guide: Preclinical Screening, ClinicalTrials, and Approval” (B. Teicher, Ed.), Humana Press Inc., Totowa(1997)). The model is rapid—the mean survival of untreated animals istypically˜7-8 days—and the administered tumor cells seed in the liverand bone marrow.

Administration of free topotecan as a single intravenous dose hadminimal effect on survival in the L1210 model (see, FIG. 3A). At thehighest dose of free topotecan, a median survival of 13 days (44% ILS)was observed. There was one long-term survivor (day 60) in this group.In contrast, a single i.v. administration of liposomal topotecan ateither 5 or 10 mg/kg resulted in 100% survival at day 60 (see, FIG. 3B).Median survival for a 1 mg/kg dose was 13 days (44% ILS) and thesurvival curve was nearly identical to that of the free topotecanadministered at 30 mg/kg-a 30-fold improvement in potency. At higherdoses (30 mg/kg) of the liposomal topotecan, toxic deaths were observed.The MTD for liposomal topotecan was 20 mg/kg in BDF-1 mice after asingle i.v. administration.

2. CT-26 Murine Colon Carcinoma. The murine CT-26 colon cell line isuseful for drug screening since it readily grows as subcutaneous solidtumors or can be administered intravenously and used as a survivalmodel. In addition, when the tumor cells are administered byintrasplenic injection, followed by splenectomy, the cells seed to theliver and give rise to an experimental metastases model that moreclosely resembles the clinical progression of colorectal cancer. Themodel has been used extensively and is described, for example, in detailelsewhere.

In the CT-26 model, administration of a single dose of topotecan had amodest impact on survival resulting in % ILS of 23-60% over the doserange 5-40 mg/kg (see, FIG. 4). Liposome encapsulated topotecan,however, was highly active at doses greater than 5 mg/kg, resulting in100% survival (8/8) at day 90. At 10 mg/kg, 87.5% survival (7/8) wasobserved at day 90; however, the tumor burden in dead animal was verylow suggesting that this animal may have died due to other factors, suchas infection related to myelosuppression. A dose response was observedfor liposomal topotecan, with the 2 mg/kg dose giving an % ILS of 54%.This was determined to be the MED and was comparable to the % ILS (58%)achieved using free topotecan at 40 mg/kg —a 20-fold increase inpotency.

3. MX-1 Human Breast Carcinoma. Mx-1 is an experimental model of humanbreast cancer and has a reported doubling time of 3.9 days NCI); in thisstudy, the median doubling time was consistently 3.6-3.7 days. The tumorcell line was derived from the primary tumor of a 29-year-old femalewith no previous history of chemotherapy and is provided by the DCTD(NCI) tumor repository as a tumor fragment that is serially passaged innude mice. Histologically, MX-1 is a poorly differentiated mammarycarcinoma with no evidence of gland formation or mucin production. MX-1was one of 3 xenograft models (MX-1, LX-1, CX-1) that comprised the NCIin vivo tumor panel and prescreen (1976-1986) for evaluating novelchemotherapeutic agents (Plowman, et al., Human tumor xenograft modelsin NCI drug development. In “Anticancer Drug Development Guide:Preclinical Screening, Clinical Trials, and Approval” (B. Teicher, Ed.),Humana Press Inc., Totowa (1997)). Since then, MX-1 has beenincorporated into a larger panel of breast tumor models (12 in total) toreflect a shift in NCI strategy from “compound-oriented” discovery to“disease-oriented” discovery.

In staged (100-300 mm³) MX-1 tumors, free topotecan exhibiteddose-dependent inhibition of tumor growth (see, FIG. 5; Table I). At thehighest dose (40 mg/kg), an optimal % T/C of 24% was obtained; whileoptimal % T/C values for 10 and 5 mg/kg were 66% and 78%, respectively.No drug-related deaths were observed and all animals gained weightthroughout the study. Liposome encapsulation of topotecan had a markedimpact on % T/C, with optimal % T/C values of 8%, −49% and −62%following a single administration of the drug at 2, 5 or 10 mg/kg,respectively. A negative % T/C value is indicative of tumor volumeregression from the original staged tumor size (100-300 mm³). Accordingto NCI guidelines, an optimal % T/C <10% is considered significantactivity, while values <42% are the minimum acceptable limits foradvancing a drug further in development (Corbett, T. et al., In vivomethods for screening and preclinical testing. In “Anticancer DrugDevelopment Guide: Preclinical Screening, Clinical Trials, and Approval”(B. Teicher, Ed.), Humana Press Inc., Totowa (1997)). Liposomeencapsulation increased the toxicity of topotecan, reducing the MTD to10 mg/kg from >40 mg/kg for free topotecan.

4. LX-1 Human Lung Carcinoma. LX-1 is an experimental model of humansmall cell lung cancer (SCLC). The tumor cell line was derived from thesurgical explant of a metastatic lesion found in a 48 year old male andis provided by the DCTD (NCI) tumor repository as a tumor fragment thatis serially passaged in nude mice. The LX-1 model was part of the NCI invivo tumor panel from 1976-1986 (Plowman, J. et al, Human tumorxenograft models in NCI drug development. In “Anticancer DrugDevelopment Guide: Preclinical Screening, Clinical Trials, and Approval”(B. Teicher, Ed.), Humana Press Inc., Totowa (1997)) and, although usedless frequently now, remains a useful xenograft model for comparativeactivity studies between free and liposomal drugs because of its rapidgrowth rate.

In general, the LX-1 model was less sensitive to the effects oftopotecan than the MX-1 model, for both free and liposome-encapsulateddrug (see, FIG. 6; Table I). Optimal % T/C values for free topotecanwere 43%, 55% and 67% for doses of 30, 10 or 5 mg/kg, respectively.Anti-tumor activity was improved through encapsulation, resulting in %T/C values of 8%, 11% and 13% for doses of 30, 10, or 5 mg/kg,respectively. Interestingly, all of the liposomal topotecan dosesexhibited similar activity. This was an early study and subsequentstudies in other models (see, FIGS. 4-6) indicate dose responsebeginning at doses <5 mg/kg. This is consistent with the observationthat camptothecin-class compounds (and presumably other antineoplasticagents) can exhibit “self-limiting” efficacy whereby, at doses above acritical threshold dose, no further activity benefits are observed(Thompson, Biochim. Biophys. Acta 1400:301-319 (1998)). This situationcould conceivably occur if the drug has limited tumor cell access or ifthe drug is acting on, and destroying, the tumor vasculature (i.e., hasanti-angiogenic activity). In both instances, a higher dose of drugwould be expected to have negligible benefit.

As observed in the L1210 study, encapsulation of topotecan enhanced thetoxicity of the drug and reduced the MTD. The MTD in tumor-bearing nudemice was 10 mg/kg (˜16% weight loss). At 30 mg/kg, 4/6 drug-relatedtoxic deaths were observed and maximum weight loss reached ˜29% (27-34%range).

D. Efficacy Studies in Murine and Human Tumor Models: Multiple DoseStudies

1. MX-1 Human Breast Carcinoma. To address the effectiveness of multipleadministration and prolonged exposure of the tumors to drug, twomultiple dose protocols were examined in MX-1 xenografts—q3dx4 and q7dx3schedules. On the q4dx3 schedule, free topotecan exhibited moderateactivity at 2.5 and 10 mg/kg/dose and minimal activity at 1.25mg/kg/dose (see, FIG. 7; Table II). Optimal % T/C values for freetopotecan on this dosing schedule were 55%, 30% and 27% for 1.25, 2.5and 10 mg/kg/dose, respectively. For the encapsulated topotecanadministered on the same dosing schedule, optimal % T/C values were−15%, −100%, −100%, and −100% for 0.5, 1.25, 2.5 and 5 mg/kg/dose,respectively. All regressed tumors were monitored for 60 days. At theend of this period, all animals treated with ≧1.25 mg/kg/dose ofliposomal topotecan were considered tumor free.

On a q7dx3 dosing schedule, little activity was observed with the freetopotecan, either a 5 or 10 mg/kg/dose (see, FIG. 8; Table II). At thesame doses, liposomal topotecan induced complete regression of thestaged tumors. However, on this dosing schedule, 10 mg/kg/dose was tootoxic and this portion of the study was halted as 6/6 toxic deaths (orcuthanasia's) were observed by day 24.

2. LX-1 Human Lung Carcinoma. Initial studies (single dose) in the LX-1model indicated that free topotecan was inactive at evaluated doses <30mg/kg and liposomal topotecan inhibited tumor growth, but did not induceregression. To improve this activity, a multiple (q7dx3) schedule wasexamined for both free and liposomal topotecan. In this instance,considerably greater activity was observed for free topotecan comparedto the single dose study and optimal % T/C values of 5 and 40 wereobtained for 30 and 10 mg/kg/dose, respectively. Liposomal topotecanalso exhibited significantly improved activity, resulting in completeregression (with subsequent re-growth) at 5 mg/kg/dose. Optimal % T/Cvalues for liposomal topotecan in this model and dosing schedule were−55, 3 and 16 for 5, 2.5, 1.25 mg/kg/day, respectively.

3. Therapeutic Index (TI) Comparisons. The therapeutic index of free andliposomal topotecan was assessed in 4 different tumor models on severaldifferent dosing schedules (see, Table 4). The assumptions anddefinitions used to generate these numbers are found in Table III. Insome instances, a true MED or MTD was not observed and was thereforeestimated mathematically based on dose response trends. For instance, anacute MTD of 40 mg/kg was observed for free topotecan administered as asingle bolus injection, but the true MTD (based on weight loss) wouldlikely be closer to 60 mg/kg if the drug was infused over 5-10 minutes.Also, complicating the analysis somewhat was the level of potency of theliposomal formulation. Significant anti-tumor activity was achieved atlow drug doses and the MED had to be estimated in certain studies. Inthese instances, a notation was made in Table 4.

In general, the increase in therapeutic index for liposomal topotecanwas relatively large for single dose administration (5, 10, 15 and18-fold, depending on the model) and decreased with increasing dosingfrequency. This is illustrated in Table 4, where the TI_(TCS)/TI_(Free)ratio was 4.7-7.5 and 3.3 for q7dx3 and q3dx4 schedules, respectively.The decrease in the TI_(TCS)/TI_(Free) ratio with more frequent dosingis consistent with preclinical and clinical studies indicating that theefficacy and toxicity of free topotecan is schedule-dependent.

TABLE 4 Relative Therapeutic Indices of Free and Liposomal Topotecan inMurine and Human Tumor Models.^(a) Tumor Model Route of InoculationDosing Schedule TI_(Free) TI_(TCS) TI_(TCS)/TI_(Free) L1210 (murineleukemia) i.v. single 1.3 (2.0)^(b) 20 15.4 (10)^(b) CT-26 (murinecolon) i.s. single 1.0 (1.5)^(b) 5.0 5 (3.3)^(b) MX-1 (human breast)s.c. single 1.4 (2.1)^(b) 25 17.9 (11.9)^(b) q3dx4 15 50^(c) 3.3 q7dx32.0 15.0^(c) 7.5 LX-1 (human lung) s.c. single 1.3 (2.0)^(b) 13.3 10.2(67)^(b) q7dx3 4.0 18.8 4.7 ^(a)based on data in Table II and III;formulas and definitions in Table IV. ^(b)obtained using an acute MTD of40 mg/kg; second value is based on an estimated MTD (body weight) ^(c)aconservative estimate that may be ~2-fold greater; difficult to assessthe MED due to high activity at low doses.

E. Discussion

Topotecan is an excellent candidate for liposome encapsulation. Briefly,topotecan is cell-cycle specific (S-phase) and activity is greatlyenhanced with prolonged exposure, topotecan exhibits rapid plasmapharmacokinetics and the drug needs to be maintained below pH 6.0 toretain biological activity. This is an ideal scenario for using arelatively non-leaky liposome formulation (such as SM:CH, 55:45) thathas an acidic aqueous core. The required acidic interior can beproduced, for example, by pH-loading or ionophore loading methodology.Here, it has been demonstrated that encapsulation of topotecan in SM/CHliposomes by the Mg-A23187 method results in dramatic enhancements inanti-tumor efficacy. Modest enhancement of toxicity was also observedfor liposomal topotecan, but this was largely offset by substantial dosereductions that achieved comparable and, in most instances, superiorefficacy relative to the free drug.

Therapeutic index (TI) is a useful parameter of drug activity, as it ismeasure of the ratio of toxicity (MTD) to biological activity (userdefined endpoint, i.e., MED, ED₅₀, or ED₈₀). In general, the lower theTI, the greater the risk of toxicity since the dose of drug required toelicit a biological effect approaches the MTD. Therapeutic index isparticularly useful for the evaluation of liposomal drugs since therelative change in TI can be used to define the benefit (or lackthereof) of encapsulation. As demonstrated herein, the TI improved from3-18 fold depending on the model and dose schedule used. Therefore, theimprovement in biological activity observed following liposomeencapsulation of topotecan more than compensates for any increases intoxicity.

Without intending to be bound by any theory, it is thought that thesignificant improvements in anti-tumor activity and the increasedtoxicity of the liposomal form of the drug result from improvedpharmacokinetics and the maintenance of the drug in the active lactoneform. In these studies, 84% of topotecan was present in plasma as thelactone species after 24 h compared to 48% lactone for free topotecanafter only 5 minutes. Moreover, when the same dose (10 mg/kg) of freeand liposomal topotecan was administered intravenously in mice, theconcentration of lactone was ˜40-fold higher at times <1 h. At 24 h, thelactone plasma concentration for liposomal drug was 5.4 μg/ml comparedto 1.5 μg/ml at 5 minutes for free drug—still 3.5-fold greater than thepeak lactone concentration for free topotecan.

TABLE I Summary of Single Dose Anti-Tumor Activity and ToxicityParameters Anti-Tumor Activity Toxicity Model Dose % T/C^(a) T-C^(b) %ILS^(c) LCK^(d) TF^(e) DRD^(f) MWL^(g) L1210 Free 5 11 0/8 0/8 + (i.v.)Free 10 22 0/8 0/8 + NCTEF-005 Free 20 33 0/8 0/8 + Free 30 44 0/8 0/8 +Free 40 55 0/8 0/8 + TCS 1 44 0/8 0/8 + TCS 5 ** 8/8 0/8 + TCS 10 ** 8/80/8 −9.7 TCS 20 ** 7/7 1/8 −14.8 TCS 30 ** 3/3 5/8 −23.4 CT-26 Free 5 310/8 0/8 + (i.s.) Free 10 23 0/8 0/8 + NCTEF-005 Free 40 58 1/8 0/8 −0.4TCS 2 54 0/8 0/8 + TCS 5 ** 8/8 0/8 −6.8 TCS 10 ** 7/8 0/8 −19.1 MX-1Free 5 78 0.2  0 0.02 0/6 0/6 + (s.c.) Free 10 66 1.4 13 0.12 0/6 0/6 +NCTEF-004 Free 40 24 4.2 35 0.35 0/6 0/6 + TCS 2 8 7.4 65 0.62 0/6 0/6 +TCS 5 −49 10.2 74 0.85 0/6 0/6 −0.4 TCS 10 −62 14.2 83 1.19 1/6 0/6−18.3 LX-1 Free 5 67 1.4  0 0.13 0/6 0/6 + (s.c.) Free 10 55 1.9  0 0.180/6 0/6 + NCTEF-003 Free 30 43 2.9  7 0.27 0/6 0/6 −1.3 TCS 5 13 7.9 300.74 0/6 0/6 −1.7 TCS 10 11 8.7 22 0.82 0/6 0/6 −15.6 TCS 30 8 9.9 220.93 0/6 4/6 −29.0 ^(a)optimal % T/C following final treatment. Negativevalue indicates tumor regression. ^(b)tumor growth delay (difference intime for treated and control tumors to reach 500 mm³). ^(c)increase inlifespan relative to untreated animals (expressed as %). ^(d)log cellkill (gross). ^(e)tumor free animals at the end of study (i e. novisible tumors or long term survivors). ^(f)drug related deaths.^(g)maximum mean weight loss per treatment group. ^(h)positive weightchange (i e. at no time did weight decrease below pre-treatment weight).**long term survivors

TABLE II Summary of Multiple Dose Anti-Tumor Activity and ToxicityParameters Anti-Tumor Activity Toxicity Model Dose % T/C^(a) T-C^(b) %ILS^(c) LCK^(d) TF^(e) DRD^(f) MWL^(g) MX-1 Free 1.25 55 2.0 20 0.17 0/60/6  +^(h) (q3dx4) Free 2.5 30 5.0 55 0.42 0/6 0/6 + NCTEF-006 Free 1027 2.5 52 0.21 1/6 0/6 + TCS 0.5 −15 23.5 157 1.96 1.6 0/6 −0.3 TCS 1.25−100 ** ** 6/6 0/6 −1.0 TCS 2.5 −100 ** ** 6/6 0/6 −11.5 TCS 5 −100 **** 6/6 0/6 −20.0 MX-1 Free 5 58 1.8 27 0.15 0/6 0/6 + (q7dx3) Free 10 612.0 ND^(i) 0/6 0/6 −0.8 NCTEF-009 TCS 5 −100 ** ** 6/6 0/6 −7.6 TCS 10−100 ND^(i) ND^(i) 6/6 6/6 −29.0 LX-1 Free 10 40 2.0 21 0.14 0/6 0/6−6.2 (q7dx3) Free 30 5 20.9 58 1.53 0/6 0/6 −8.8 NCTEF-007 TCS 1.25 1610.8 54 0.79 0/6 0/6 −7.7 TCS 2.5 3 23.2 79 1.70 0/6 0/6 −7.3 TCS 5 −5530.2 100 2.22 0/6 0/6 −10.5 LX-1 Free 10 28 4.4 41 0/6 0/6 −3.6 (q7dx3)Free 30 9 25 72 0/6 2/6 −16.4 NCTEF-011 TCS 7.5 ND^(i) ND^(i) ND^(i) 0/66/6 >−30 TCS 0.75 27 11.2 50 0/6 0/6 −1.3 ^(a)optimal % T/C followingfinal treatment. Negative value indicates tumor regression. ^(b)tumorgrowth delay (difference in time for treated and control tumors to reach500 mm3). ^(c)increase in lifespan relative to untreated animals(expressed as %) ^(d)log cell kill (gross). ^(e)tumor free animals atthe end of study (i.e. no visible tumors or long term survivors).^(f)drug related deaths. ^(g)maximum mean weight loss per treatmentgroup. ^(h)positive weight change (i.e. at no time did weight decreasebelow pre-treatment weight). ^(i)not determined; toxic deaths in theliposome-encapsulated group. **“cures”; no visible tumors by day 60.

TABLE III Definitions and Formulas for Toxicity and Anti-Tumor ActivityParameters DRD Drug-related death. A death was considered drug-relatedif the animal died or was euthanized within 15 days following the finaltreatment with drug AND its tumor weight was less than the lethal burdenon control mice, or its weight loss was greater than 20% that of thecontrol animals. GI₅₀ The concentration of drug that causes 50% growthinhibition in a population of cells in vitro. The NCI renamed the IC₅₀parameter to emphasize the correction for cell count at time zero.Therefore, the formula is: GI₅₀ = (T − T_(o))/(C − T_(o)) × 100 = 50 Tand T_(o) are the optical densities at 48 and 0 h, respectively; C isthe control (cell count) optical density at 0 h. % ILS Increase inlifespan (in percent). For survival models this is calculated using themedian survival times for the treated (T_(treat)) and control (T_(cont))animals, according to: (T_(treat) − T_(cont))/T_(cont) × 100 For thesolid tumor models, the time for tumors to reach 2000 mm³ (~10% of bodyweight) was used as an ethical cutoff instead of median survival. LCKLog cell kill (gross). This parameter estimates the number of log₁₀units of cells killed at the end of treatment, according to the formula:(T − C) × 0.301/median doubling time Net log cell kill can be calculatedby subtracting the duration of treatment from the tumor growth delay (T− C) parameter as follows: [(T − C) − duration of treatment] ×0.301/median doubling time A log cell kill of 0 indicates that the cellpopulation at the end of treatment is the same as it was at the onset oftreatment. However, a log cell kill of 4, for example, indicates a99.99% reduction in the initial cell population. MBWL Maximum bodyweight loss (in percent). The animals are weighed prior to the firstadministration of the drug (Wi) and on various days during the study(Wd). The percent change in body weight is calculated by: MBWL = (W_(d)− W₁)/W_(i) × 100 MED Minimum effective dose. This is a somewhatarbitrary parameter. For these studies we have defined the MED as thelowest dose achieving an optimal % T/C ≦ 40 (for solid tumor models) ora % ILS of 40–60% (for survival models). MTD Maximum tolerated dose.Dose of drug that results in a MBWL of ≦20%. % T/C Optimal ratio oftreated vs control tumors obtained following the first course oftreatment. These values are obtained by subtracting the median tumorweight on the first day of treatments (T_(i) or C₁) from the tumorweights on each observation day according to the following formula: %T/C = (Δ T/Δ C) × 100, where Δ T > 0, or % T/C = (Δ T/T₁) × 100, where ΔT < 0 According to NCI activity criteria, the following scoring systemapplies (Plowman, et al., Human tumor xenograft models in NCI drugdevelopment. In “Anticancer Drug Development Guide: PreclinicalScreening, Clinical Trials, and Approval” (B. Teicher, Ed.), HumanaPress Inc., Totowa (1997)[22]: 0 = inactive, % T/C > 40 1 = tumorinhibition, % T/C range 1–40 2 = tumor stasis, % T/C range 0 to −40 3 =tumor regression, % T/C range −50 to −100 4 = % T/C range −50 to −100and > 30% tumor-free mice TGD Tumor growth delay (also represented as T− C). This parameter expresses the difference in time (in days) fortreated and control tumors to attain an arbitrary size (typically 500 or1000 mm³). TI Therapeutic index. Therapeutic index is the ratio of atoxicity parameter (i.e. LD₅₀, LD₁₀, MTD) and a biological activityparameter (i.e. ED₅₀-the dose that causes a defined biological responsein 50% of the treatment group). In general, TI describes the margin ofsafety for a drug. For animal model studies this is traditionallydescribed by the formula: TI = LD₅₀/ED₅₀ However, since it is no longerethically permissible to perform LD₅₀ studies, we have definedtherapeutic index for these studies as: TI = MTD/MED

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the fall scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications and publications, areincorporated herein by reference for all purposes.

1. A liposomal formulation, said liposomal formulation comprising aliposome having vinorelbine in solution and precipitated vinorelbine,wherein the precipitated vinorelbine in said liposome is at least 50% ofthe total vinorelbine, wherein said liposome comprises sphingomyelin andcholesterol at a ratio in the range of about 75/25 mol %/mol %sphingomyelinl/cholesterol to about 30/50 mol %/mol %sphingomyelinl/cholesterol, and wherein the ratio of said vinorelbine tolipid is 0.2-0.3:1 (w/w).
 2. The liposomal formulation of claim 1,wherein said liposome comprises sphingomyelin and cholesterol in a 55:45molar ratio.
 3. The liposomal formulation of claim 1, wherein saidliposome comprises sphingomyelin and cholesterol in a 50:50 molar ratio.4. The liposomal formulation of claim 1, wherein the ratio of saidvinorelbine to said lipid is about 0.3:1 (w/w).
 5. A liposomalformulation, said liposomal formulation comprising a liposome havingvinorelbine in solution and precipitated vinorelbine, wherein theprecipitated vinorelbine in said liposome is at least 50% of the totalvinorelbine, wherein said liposome comprises sphingomyelin andcholesterol at a ratio of about 55/45 mol %/mol %sphingomyelin/cholesterol, and wherein the ratio of said vinorelbine tolipid is about 0.3:1 (w/w).